Adaptor protein FRS2 and related products and methods

ABSTRACT

The present invention relates generally to a newly identified adaptor protein FRS2 and related products and methods. FRS2 links protein kinases to activating partners in cells. The invention also relates to nucleic acid molecules encoding portions of FRS2, nucleic acid vectors containing FRS2 related nucleic acid molecules, recombinant cells containing such nucleic acid vectors, polypeptides purified from such recombinant cells, antibodies to such polypeptides, and methods of identifying compounds that enhance or block FRS2 interactions with natural binding partners. Also disclosed are methods for diagnosing abnormal conditions in an organism with FRS2 related molecules or compounds.

RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.60/032,093, filed Dec. 3, 1996, entitled “Adaptor Protein FRS2 andRelated Products and Methods” by Kouhara et al. which is incorporatedherein by reference in its entirety, including any drawings.

INTRODUCTION

The present invention relates generally to a newly identified adaptorprotein FRS2 and related products and methods. FRS2 links proteinkinases to activating partners.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedto aid in understanding the invention, but is not admitted to describeor constitute prior art to the invention.

Cellular signal transduction is a fundamental mechanism wherebyextracellular stimuli are relayed to the interior of cells andsubsequently regulate diverse cellular processes. One of the keybiochemical mechanisms of signal transduction involves the reversiblephosphorylation of proteins. Phosphorylation of polypeptides regulatesthe activity of mature proteins by altering their structure andfunction. Phosphate most often resides on the hydroxyl moiety (—OH) ofserine, threonine, or tyrosine amino acids in proteins.

Enzymes that mediate phosphorylation of cellular effectors generallyfall into two classes. The first class consists of protein kinases whichtransfer a phosphate moiety from adenosine triphosphate to proteinsubstrates. The second class consists of protein phosphatases whichhydrolyze phosphate moieties from phosphoryl protein substrates. Theconverse functions of protein kinases and protein phosphatases balanceand regulate the flow of signals in signal transduction processes.

Protein kinases are generally divided into two groups—receptor andnon-receptor type proteins. Receptor protein kinases straddle the cellmembrane and harbor an extracellular region, a transmembrane region, andan intracellular region. Non-receptor protein kinases exist within thecell and harbor a catalytic region attached to other functional regionsthat can localize the protein kinase to different regions in the cell.

Protein kinases are also typically divided into three classes based uponthe amino acids they act upon. Some phosphorylate serine or threonineonly, some phosphorylate tyrosine only, and some phosphorylate serine,threonine, and tyrosine.

Many protein kinases, particularly receptor protein kinases, function bybinding adaptor proteins. Adaptor proteins link the protein kinase toother proteins that cause a cellular reaction to a protein kinasesignal. The epidermal growth factor receptor (EGFR), for example,phosphorylates itself upon binding the EGF ligand. The resultingphosphate moieties on the EGFR intracellular region bind adaptorproteins such as Grb-2. Grb-2 then binds a guanine nucleotide exchangefactor protein (Sos), which thereby activates Ras. Ras consequentlyactivates the mitogen activated protein kinase (MAPK) cascade, whichcauses cellular proliferation or differentiation. Thus, Sos and Ras aredirect activating partners of EGFR while members of the MAPK cascade areindirect activating partners of EGFR.

Multiple adaptor proteins harbor domains that directly bind to thephosphate moieties on receptor protein kinase. Grb-2, for example,harbors a Src homology 2 domain (SH2 domain) that tightly bindsphosphotyrosine moieties within EGFR and other receptor protein kinases.Pawson and Schlessinger, 1993, Current Biol. 3:434-442. Other adaptorproteins, such as IRS-1, can bind phosphotyrosine moieties of receptorprotein kinases via a phosphoryl tyrosine binding domain (PTB domain).Gustafson et al., 1995, Mol. Cell. Biol. 15:2500-2508. Adaptors such asShc harbor both SH2 and PTB domains. Blaikie et al., 1994, J. Biol.Chem. 269:32031-32034. Some adaptor proteins, such as SNT-like proteins,harbor unidentified phosphotyrosine binding domains because theirnucleotide and amino acid sequences are unknown. Wang et al., 1996,Oncogene 13:721-729.

It has become evident that receptor protein kinases other than EGFR,such as the fibroblast growth factor receptor protein kinase (FGFR),stimulate the MAPK cascade without directly binding Grb-2. Nakafuku etal., 1992, J. Biol. Chem. 267:22963-22966. Scientists are thereforesearching for adaptor proteins that link protein kinases to theiractivating partners to determine the mechanism of activation for theseprotein kinases. Adaptor proteins involved in protein kinase activationmechanisms are drug targets as compounds that can enhance or abrogatethe interactions between the proteins in a protein kinase activationmechanism could potentially prevent and even treat abnormal conditionsin organisms caused by altered protein kinase function. Examples ofabnormal conditions caused by altered protein kinase function are cancerand other cell proliferative disorders such as arthritis,glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic microangiopathy syndromes, transplant rejection,glomerulopathies, hepatic cirrhosis, ocular diseases such as diabeticretinopathy, and restenosis.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid molecules encoding a newlyidentified protein kinase adaptor protein named FRS2. The invention alsorelates to nucleic acid molecules encoding portions of the full lengthprotein, nucleic acid vectors harboring such nucleic acid molecules,cells containing such nucleic acid vectors, purified polypeptidesencoded by such nucleic acid molecules, antibodies to such proteins andpolypeptides, and methods of identifying compounds that enhance or blockinteractions of FRS2 with natural binding partners. Also disclosed aremethods for diagnosing abnormal conditions in an organism with FRS2related molecules or compounds. The nucleic acid molecules, nucleic acidvectors, host cells, polypeptides, and antibodies may be produced usingthe information provided herein in conjunction with well known andstandard techniques used currently in the art.

FRS2 (Fibroblast Growth Factor Receptor Protein Kinase substrate 2)regulates growth factor stimulation of cellular differentiation andcellular proliferation by linking stimulated fibroblast growth factorreceptor (FGFR) to the Ras/MAPK cascade via the Grb-2/Sos complex.Various treatments of cell proliferative disorders and celldifferentiation disorders are therefore provided based on the discoveryof FRS2.

The FRS2 adaptor protein of the invention is isolated from human NIH 3T3cells. The invention also relates to closely related adaptor proteinspreferably from mammalian tissue and more preferably from human tissue.The term “closely related” refers to greater than 50% amino acididentity or a similar three dimensional structure. The term “amino acididentity” is described herein.

Thus in a first aspect, the invention features an isolated, enriched, orpurified nucleic acid molecule encoding a FRS2 polypeptide.

The term “isolated”, in reference to nucleic acid molecules, indicatesthat a naturally occurring sequence has been removed from its normalcellular environment. Thus, the sequence may be in a cell-free solutionor placed in a different cellular environment. The term does not implythat the sequence is the only nucleotide chain present, but that it isessentially free (about 90-95% pure at least) of non-nucleotide materialsuch as chromosomal DNA or proteins.

The term “enriched”, in reference to nucleic acid molecules, means thatthe specific DNA or RNA sequence constitutes a significantly higherfraction (2-5 fold) of the total DNA or RNA present in the cells orsolution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. A person skilled in the art couldenrich a nucleic acid mixture by preferentially reducing the amount ofother DNA or RNA present, or preferentially increasing the amount of thespecific DNA or RNA, or both. However, nucleic acid molecule enrichmentdoes not imply that there is no other DNA or RNA present, the term onlyindicates that the relative amount of the sequence of interest has beensignificantly increased. The term “significantly” qualifies “increased”to indicate that the level of increase is useful to the personperforming the recombinant DNA technique, and generally means anincrease relative to other nucleic acids of at least 2 fold, or morepreferably at least 5 to 10 fold or more. The term also does not implythat there is no DNA or RNA from other sources. Other DNA may, forexample, comprise DNA from a yeast or bacterial genome, or a cloningvector. In addition, levels of mRNA may be naturally increased relativeto other species of mRNA when working with viral infection or tumorgrowth techniques. The term “enriched” is meant to cover only thosesituations in which a person has intervened to elevate the proportion ofthe desired nucleic acid.

Many methods of recombinant nucleic acid manipulation require that thesemolecules are in a purified form. The term “purified”, in reference tonucleic acid molecules does not require absolute purity (such as ahomogeneous preparation); instead, it represents an indication that thesequence is relatively more pure than in its cellular environment(compared to the natural level this level should be at least 2-5 foldgreater, e.g., in terms of mg/ml). The claimed DNA molecules obtainedfrom clones could be obtained directly from total DNA or from total RNA.cDNA clones are not naturally occurring, but rather are preferablyobtained via manipulation of a partially purified, naturally occurringsubstance (messenger RNA). The construction of a cDNA library from mRNAinvolves the creation of a synthetic substance (cDNA). Individual cDNAclones can be isolated from the synthetic library by clonal selection ofthe cells carrying the cDNA library. Thus, the process which includesthe construction of a cDNA library from mRNA and isolation of distinctcDNA clones yields an approximately 10⁶-fold purification of the nativemessage. Thus, purification of at least one order of magnitude,preferably two or three orders, and more preferably four or five ordersof magnitude is favored in these techniques.

The term “nucleic acid molecule” describes a polymer ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acidmolecule may be isolated from a natural source by cDNA cloning orsubtractive hybridization or synthesized manually. The nucleic acidmolecule may be synthesized manually by the triester synthetic method orby using an automated DNA synthesizer.

The term “cDNA cloning” refers to hybridizing a small nucleic acidmolecule, a probe, to genomic cDNA that is bound to a membrane. Theprobe hybridizes (binds) to complementary sequences of cDNA.

The term “complementary” describes two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymidine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. Thus a “complement” of a nucleic acid molecule isa molecule containing adenine instead of thymine, thymine instead ofadenine, cytosine instead guanine, and guanine instead of cytosine.Because the complement contains a nucleic acid sequence that formsoptimal interactions with the parent nucleic acid molecule, such acomplement binds with high affinity to its parent molecule.

The term “hybridize” refers to a method of interacting a nucleic acidprobe with a DNA or RNA molecule in solution or on a solid support, suchas cellulose or nitrocellulose. If a nucleic acid probe binds to the DNAor RNA molecule with high affinity, it is said to “hybridize” to the DNAor RNA molecule. As mentioned above, the strength of the interactionbetween the probe and its target can be assessed by varying thestringency of the hybridization conditions. Various low or highstringency hybridization conditions may be used depending upon thespecificity and selectivity desired. Stringency is controlled by varyingsalt or denaturant concentrations. Under stringent hybridizationconditions only highly complementary nucleic acid sequences hybridize.Preferably, such conditions prevent hybridization of nucleic acidshaving one or two mismatches out of 20 contiguous nucleotides.

cDNAs are molecules that may be reverse-transcribed from fragments ofmessage RNA from a genomic source. These fragments form a cDNA libraryof nucleic acid molecules. cDNA libraries are constructed from naturalsources such as mammalian blood, semen, or tissue.

The term “subtractive hybridization” refers to a method similar to cDNAcloning except that cDNA prepared from mRNA in unstimulated cells isadded to mRNA in stimulated or different types of cells. cDNA/mRNA canthen be precipitated to enrich the mRNA specific to the stimulationsignal or different cell type.

The term “FRS2 polypeptide” refers to a polypeptide having an amino acidsequence preferably of at least 400 contiguous amino acids, morepreferably of at least 450 contiguous amino acids, or most preferably ofat least 508 contiguous amino acids set forth in FIG. 1A, or issubstantially similar to such a sequence. A sequence that issubstantially similar will preferably have at least 90% identity (morepreferably at least 95% and most preferably 99-100%) identity to theamino acid sequence of FIG. 1A. FRS2 polypeptides preferably have Grb-2binding activity and fragments of the full length FRS2 sequence havingsuch activity may be identified using techniques well known in the art,such as sequence comparisons and assays such as those described in theexamples herein.

“Identity” refers to a property of sequences that measures theirsimilarity or relationship. Identity is measured by dividing the numberof identical residues by the total number of residues and multiplyingthe product by 100. Thus, two copies of exactly the same sequence have100% identity, but sequences that are less highly conserved and havedeletions, additions, or replacements may have a lower degree ofidentity. Those skilled in the art will recognize that several computerprograms are available for determining sequence identity. Such programsare generally able to achieve maximum alignment by ignoring deletions oradditions that would otherwise alter the calculation of the percentageof identity between two sequences.

A preferred embodiment concerns nucleic acid molecules relating to FRS2enriched, isolated, or purified from a mammalian source. These nucleicacid molecules can be isolated from, among other sources, blood, semen,or tissue.

The term “mammalian” refers to such organisms as mice, rats, rabbits,goats, preferably monkeys and apes, and more preferably humans. Althoughthe FRS2 nucleic acid molecule of FIG. 1A is isolated from NIH 3T3cells, current recombinant DNA techniques can readily elucidate arelated nucleic acid molecule in other tissues.

Another preferred embodiment concerns an isolated nucleic acid moleculerelating to FRS2 that encodes at least twelve contiguous amino acids ofthe amino acid sequence set forth in FIG. 1A. Preferably at least 3, 5,10, 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500 or 508contiguous amino acids of the FRS2 sequence are encoded. This preferredembodiment of the invention is achieved by applying routine recombinantDNA techniques known to those skilled in the art.

Another aspect of the invention features a nucleic acid probe that candetect nucleic acid molecules encoding a FRS2 polypeptide in a sample.

The term “nucleic acid probe” refers to a nucleic acid molecule that iscomplementary to and can bind a nucleic acid sequence encoding the aminoacid sequence substantially similar to that set forth in FIG. 1A.

The nucleic acid probe or its complement encodes any one of the aminoacid molecules set forth in the invention. Thus the nucleic acid probecan encode at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 200, 300,400, 500 or 508 contiguous amino acids of the full-length sequence setforth in FIG. 1A.

The nucleic acid probe can be labeled with a reporter molecule ormolecules. The term “reporter molecule” refers to a molecule that isconjugated to the nucleic acid probe or is contained within the nucleicacid probe. The reporter molecule allows the detection of the probe bymethods used in the art. Reporter molecules are chosen from, but limitedto, the group consisting of an enzyme, such as a peroxidase, aradioactive element, or an avidin molecule.

A nucleic acid probe, whether labeled or unlabeled, should hybridize toa complement in a sample.

The nucleic acid probe can be a nucleic acid molecule encoding aconserved or unique region of amino acids. These nucleic acid moleculesare useful as hybridization probes to identify and clone additionalpolypeptides relating to FRS2.

The term “conserved nucleic acid regions”, refers to regions present intwo or more nucleic acid molecules encoding a FRS2 polypeptide, to whicha particular nucleic acid sequence can hybridize under lower stringencyconditions. Examples of lower stringency conditions suitable forscreening nucleic acid molecules are provided in Abe, et al. J. Biol.Chem. 19:13361 (1992) (hereby incorporated by reference herein in itsentirety, including any drawings). Preferably, conserved regions differby no more than 5 out of 20 nucleotides. As mentioned above, proteintyrosine kinases share conserved regions in their extracellular andcatalytic domains.

The term “unique nucleic acid region” concerns a sequence present in afull length nucleic acid coding for a FRS2 polypeptide that is notpresent in a sequence coding for any other naturally occurringpolypeptide. Such regions preferably comprise 30 or 45 contiguousnucleotides present in the full length nucleic acid sequence encoding aFRS2 polypeptide. In particular, a unique nucleic acid region ispreferably of mammalian origin.

Methods for using the probes include detecting the presence or amount ofFRS2 RNA in a sample by contacting the sample with a nucleic acid probeunder conditions such that hybridization occurs. The nucleic acid duplexformed between the probe and a nucleic acid sequence coding for a FRS2polypeptide may be used in the identification of the sequence of thenucleic acid detected (for example see, Nelson et al., in NonisotopicDNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed.,1992) hereby incorporated by reference herein in its entirety, includingany drawings). Kits for performing such methods may be constructed toinclude a container holding a nucleic acid probe.

In yet another aspect, the invention relates to a nucleic acid vectorcomprising a nucleic acid molecule encoding a FRS2 polypeptide and apromoter element effective to initiate transcription in a host cell.

The term “nucleic acid vector” relates to a single or double strandedcircular nucleic acid molecule that can be transfected or transformedinto cells and replicate independently or within the host cell genome. Acircular double stranded nucleic acid molecule can be cut and therebylinearized upon treatment with restriction enzymes. An assortment ofvectors, restriction enzymes, and the knowledge of the nucleotidesequences that the restriction enzymes operate upon are readilyavailable to those skilled in the art. A nucleic acid molecule of theinvention can be inserted into a vector by cutting the vector withrestriction enzymes and ligating the two pieces together.

Many techniques are available to those skilled in the art to facilitatetransformation or transfection of the expression construct into aprokaryotic or eukaryotic organism. Sambrook, Fritsch, Maniatis, 1989,“Molecular Cloning”, Cold Spring Harbor Laboratory Press, United States.The terms “transformation” and “transfection” refer to methods ofinserting an expression construct into a cellular organism. Thesemethods involve a variety of techniques, such as treating the cells withhigh concentrations of salt, an electric field, or detergent, to renderthe host cell outer membrane or wall permeable to nucleic acid moleculesof interest.

The term “promoter element” describes a nucleotide sequence that isincorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. The promoterelement precedes the 5′ end of the FRS2 nucleic acid molecule such thatthe latter is transcribed into mRNA. Host cell machinery then translatesmRNA into a polypeptide.

Those skilled in the art would recognize that a nucleic acid vector cancontain many other nucleic acid elements besides the promoter elementand the FRS2 nucleic acid molecule. These other nucleic acid elementsinclude, but are not limited to, origins of replication, ribosomalbinding sites, nucleic acid sequences encoding drug resistance enzymesor amino acid metabolic enzymes, and nucleic acid sequences encodingsecretion signals, periplasm or peroxisome localization signals, orsignals useful for polypeptide purification.

A nucleic acid vector can be useful for identifying natural bindingpartners of FRS2 polypeptides.

The term “natural binding partners” refers to polypeptides that bind toFRS2 and play a role in propagating a signal in a signal transductionprocess. The term “binding partner” also refers to a polypeptide thatbinds to FRS2 within a cellular environment with high affinity. Highaffinity represents an equilibrium binding constant on the order of 10⁻⁶M. However, a natural binding partner can also transiently interact witha FRS2 polypeptide and chemically modify it. FRS2 natural bindingpartners are chosen from a group consisting of, but not limited to, srchomology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine bindingdomains (PTB and PH domains), guanine nucleotide exchange factors, andreceptor and non-receptor protein kinases or protein phosphatases. Aknown natural binding partner of FRS2 is the SH2 containing Grb-2.

Methods are readily available in the art for identifying natural bindingpartners of polypeptides of interest by screening cDNA librariesincluded in one nucleic acid vector with a nucleic acid moleculeencoding the desired polypeptide in another expression construct. Vojteket al., 1993, Cell 74:205-214. These techniques often utilize two halvesof a transcription factor, one of which is fused to a polypeptideencoded by the cDNA library, and the other or which is fused to thepolypeptide of interest. Interactions between a polypeptide encoded bythe cDNA library and the polypeptide of interest are detected when theirinteraction concomitantly brings together the two halves of thetranscription factor and activates a gene that reports the interaction.Any of the nucleic molecules encoding FRS2 polypeptide can be readilyincorporated into a nucleic acid vector used in such a screeningprocedure by utilizing standard recombinant DNA techniques in the art.

Another aspect of the invention relates to a recombinant cell or tissuecomprising a nucleic acid molecule encoding a FRS2 polypeptide.

The term “recombinant” refers to an organism that has a new combinationof genes or nucleic acid molecules. A new combination of genes ornucleic acid molecules can be introduced to an organism using a widearray of nucleic acid manipulation techniques available to those skilledin the art.

The recombinant cell can be a eukaryotic or prokaryotic organism.

The term “eukaryote” refers to an organism comprised of cells thatcontain a nucleus. Eukaryotes are differentiated from “prokaryotes”which do not house their genomic DNA inside a nucleus. Prokaryotesinclude unicellular organisms such as bacteria while eukaryotes arerepresented by yeast, invertebrates, and vertebrates.

The term “organism” relates to any living being comprised of at leastone cell. An organism can be as simple as one eukaryotic cell or ascomplex as a mammal.

The recombinant cell can harbor a nucleic acid vector that isextragenomic. The term “extragenomic” refers to a nucleic acid vectorwhich does not insert into the cell genome. Many nucleic acid vectorsare designed with their own origins of replication allowing them toutilize the recombinant cell replication machinery to copy and propagatethe vector nucleic acid sequence. These vectors are small enough thatthey are not likely to harbor nucleic acid sequences homologous togenomic sequences of the recombinant cell. Thus these vectors replicateindependently of the host genome and do not recombine with or integrateinto the genome.

A recombinant cell can harbor a portion of a nucleic acid vector in anintragenomic fashion. The term “intragenomic” defines a nucleic acidconstruct that is incorporated within the cell genome. Multiple nucleicacid vectors available to those skilled in the art contain nucleic acidsequences that are homologous to nucleic acid sequences in a particularorganism's genomic DNA. These homologous sequences will result inrecombination events that integrate portions of the vector into thegenomic DNA. Those skilled in the art can control which nucleic acidsequences of the vector are integrated into the cell genome by flankingthe portion to be incorporated into the genome with homologous sequencesin the vector.

Yet another aspect of the invention features an isolated, enriched, orpurified FRS2 polypeptide.

The term “isolated”, in reference to a polypeptide, describes a polymerof amino acids conjugated to one other, including polypeptides that areisolated from a natural source or that are synthesized. In certainaspects longer polypeptides are preferred, such as those with most ofthe contiguous amino acids set forth in FIG. 1A.

The isolated polypeptides of the present invention are unique in thesense that they are not found in a pure or separated state in nature.Use of the term “isolated” indicates that a naturally occurring sequencehas been removed from its normal cellular environment. Thus, thesequence may be in a cell-free solution or placed in a differentcellular environment. The term does not imply that the sequence is theonly amino acid chain present, but that it is essentially free (about90-95% pure at least) of non-amino acid material naturally associatedwith it.

The term “enriched”, in reference to a polypeptide, defines a specificamino acid sequence constituting a significantly higher fraction (2-5fold) of the total of amino acids present in the cells or solution ofinterest than in normal or diseased cells or in the cells from which thesequence was separated. A person skilled in the art can preferentiallyreduce the amount of other amino acid sequences present, orpreferentially increase the amount of specific amino acid sequences ofinterest, or both. However, the term “enriched” does not imply thatthere are no other amino acid sequences present. Enriched simply meansthe relative amount of the sequence of interest has been significantlyincreased. The term “significant” indicates that the level of increaseis useful to the person making such an increase. The term also means anincrease relative to other amino acids of at least 2 fold, or morepreferably at least 5 to 10 fold, or even more. The term also does notimply that there are no amino acid sequences from other sources. Othersource amino acid sequences may, for example, comprise amino acidsequences from a host organism. “Enriched” is meant to cover only thosesituations in which a person has intervened to elevate the proportion ofthe desired amino acid sequence.

The term “purified”, in reference to a polypeptide, does not requireabsolute purity (such as a homogeneous preparation); instead, itrepresents an indication that the amino acid sequence is relatively morepure than in a cellular environment. The concentration of the preferredamino acid sequence should be at least 2-5 fold greater (in terms ofmg/ml) than its concentration in a cellular environment. Purification ofat least one order of magnitude, preferably two or three orders, andmore preferably four or five orders of magnitude is preferred. Thesubstance is preferably free of contamination, as indicated by puritylevels of 90%, 95%, or 99%.

A preferred embodiment relates to a FRS2 polypeptide that is a uniquefragment of a FRS2 polypeptide.

The term “unique fragment” refers to a stretch of contiguous amino acidsin FRS2 that is of a different sequence than another adaptor protein.From the sequence alignment between FRS2 and a closely related region ofthe adaptor protein IRS-1 indicates that the two sequences share onlytwo contiguous amino acids. Therefore at least 3, 5, 10, 15, 20, 25, 30,35, 40, 50, 100, 200, 300, 400, 500 or 508 contiguous amino acids of thefull-length amino acid sequence of FRS2 are unique to FRS2.

The FRS2 polypeptide can be isolated, enriched, or purified from aprokaryotic or eukaryotic recombinant cell. A eukaryotic cell can arisefrom organisms including mammals. Multiple standard techniques areavailable to those skilled in the art to facilitate isolation,enrichment, or purification of a polypeptide from recombinant cells.These methods typically include lysing the recombinant cells andseparating the polypeptide of interest from the rest of the cellpolypeptides, nucleic acids, and fatty acid-based material usingstandard chromatography techniques known in the art.

In the context of the present invention, isolation, enrichment, orpurification of polypeptides is attained by techniques that provideyields of FRS2 that can be visualized on a nitrocellulose membrane byPonceau-S staining or can be visualized on a sodium dodecyl sulfatepolyacrylamide gel by Coomassie or silver staining.

Another aspect of the invention features an antibody, that is monoclonalor polyclonal, or an antibody fragment having specific binding affinityto a FRS2 polypeptide.

Antibodies or antibody fragments are polypeptides which contain regionsthat can bind other polypeptides. The term “specific binding affinity”describes an antibody that binds to a FRS2 polypeptide with greateraffinity than it binds to other polypeptides under specified conditions.

The term “polyclonal” refers to a mixture of antibodies with specificbinding affinity to a FRS2 polypeptide, while the term “monoclonal”refers to one type of antibody with specific binding affinity to a FRS2polypeptide. Although a monoclonal antibody binds to one specific regionon a FRS2 polypeptide, a polyclonal mixture of antibodies can bindmultiple regions of a FRS2 polypeptide.

The term “antibody fragment” refers to a portion of an antibody, oftenthe hypervariable region and portions of the surrounding heavy and lightchains, that displays specific binding affinity for a particularmolecule. A hypervariable region is a portion of an antibody thatphysically binds to the polypeptide target.

Antibodies or antibody fragments having specific binding affinity to aFRS2 polypeptide may be used in methods for detecting the presenceand/or amount of a FRS2 polypeptide in a sample by probing the samplewith the antibody under conditions suitable for FRS2-antibodyimmunocomplex formation and detecting the presence and/or amount of theantibody conjugated to the FRS2 polypeptide. Diagnostic kits forperforming such methods may be constructed to include antibodies orantibody fragments specific for FRS2 as well as a conjugate of a bindingpartner of the antibodies or the antibodies themselves.

An antibody or antibody fragment with specific binding affinity to aFRS2 polypeptide can be isolated, enriched, or purified from aprokaryotic or eukaryotic organism. Routine methods known to thoseskilled in the art enable production of antibodies or antibodyfragments, in both prokaryotic and eukaryotic organisms. Purification,enrichment, and isolation of antibodies, which are polypeptidemolecules, are described above.

Another aspect of the invention features a hybridoma which produces anantibody having specific binding affinity to a FRS2 polypeptide. A“hybridoma” is an immortalized cell line which is capable of secretingan antibody, for example an antibody with specific binding affinity toFRS2.

Another aspect of the invention features an isolated, enriched, orpurified nucleic acid molecule comprising a nucleotide sequence that:(a) encodes a polypeptide having the full length amino acid sequence setforth FIG. 1A; (b) the complement of the nucleotide sequence of (a); (c)hybridizes under highly stringent conditions to the nucleotide moleculeof (a) and encodes a naturally occurring FRS2 protein; (d) a FRS2polypeptide having the full length amino acid sequence of sequence setforth in FIG. 1A except that it lacks one or more of the followingsegments of amino acid residues 1-10, 11-152, or 153-508; (e) thecomplement of the nucleic acid sequence of (d); (f) a polypeptide havingthe amino acid sequence set forth in FIG. 1A from amino acid residues1-10, 11-152, 153-508; (g) the complement of the nucleic acid sequenceof (f); (h) encodes a polypeptide having the full length amino acidsequence set forth in FIG. 1A except that it lacks one or more of thedomains selected from the group consisting of a myristylation region, aphosphotyrosine binding region, and a C-terminal region; (i) thecomplement of the nucleic acid sequence of (h); (j) encodes apolypeptide of (a), (d), or (f) containing one or both of the mutationstyrosine 349 to phenylalanine or tyrosine 392 to phenylalanine; or (k)the complement of the nucleic acid sequence of (j).

The term “myristylation region” refers to a portion of the full lengthFRS2 amino acid sequence that harbors a myristoyl fatty acid moiety.This region preferably spans from the amino acids 1 through 10.

The term “phosphotyrosine binding region” refers to a portion of theFRS2 amino acid molecule that can bind to a phosphotyrosine moietywithin another protein or polypeptide. This region preferably spans fromamino acids 11 through 152.

The term “C-terminal domain” refers to a portion of FRS2 that begins atthe end of the phosphotyrosine binding region to the carboxy terminalend of FRS2. This region preferably spans from amino acids 153 to 508.

Functional regions of FRS2 may be identified by aligning the amino acidsequence of FRS2 with amino acid sequences of other polypeptides withknown functional regions. If regions of FRS2 share high amino acididentity with the amino acid sequences of known functional regions, thenFRS2 can be determined to contain these functional regions by thoseskilled in the art. The functional regions can be determined, forexample, by using computer programs and sequence information availableto those skilled in the art.

Other functional regions of signal transduction molecules that may existin the FRS2 amino acid sequence include, but are not limited to,proline-rich regions or phosphoryl tyrosine regions. These regions caninteract with natural binding partners such as SH2 or SH3 domains ofother signal transduction molecules. Examples of two potential SH2binding regions of FRS2 are tyrosine 349 and 392. If these tyrosines arephosphorylated in the cell, SH2 containing proteins, such as Grb-2, canbind FRS2 at these sites. Mutating these tyrosines to phenylalanine willabrogate the binding of any SH2 containing proteins at these mutatedsites.

In yet another aspect, the invention includes a nucleic acid vectorcontaining a nucleic acid molecule described above.

Another aspect of the invention relates to a recombinant cell or tissuethat contains a nucleic acid molecule described above.

In yet another aspect, the invention features a method of identifyingcompounds capable of blocking or enhancing interactions between FRS2 andnatural binding partners. These compounds are potentially useful fordiagnosing, preventing, or treating abnormal conditions in an organism.The method consists of the following steps: (a) adding a compound tocells containing a FRS2 polypeptide; and (b) detecting a change ininteractions between FRS2 and natural binding partners.

The term “compound” includes small organic molecules including, but notlimited to, oxindolinones, quinazolines, tyrphostins, quinoxalines, andextracts from natural sources.

The term “interactions” refers to FRS2 binding to natural bindingpartners. The invention discloses that FRS2 binds to Grb-2. Thusinteractions are formed between FRS2 amino acids and the amino acids ofGrb-2. An interaction between two molecules preferably relates to thetwo molecules binding to one another and forming a complex.

The term “blocking interactions” refers to decreasing the concentrationof a complex formed between two molecules. The decrease in theconcentration of the complex can be achieved by decreasing theprobability that the two molecules form a complex by binding a compoundto one of them. A compound that binds with high affinity to one of themolecules in the complex can decrease the probability that a complexforms between the molecules if the compound decreases the likelihoodthat amino acids between the two molecules can form favorableinteractions with one another.

The term “enhancing interactions” refers to increasing the concentrationof a complex formed between two molecules. The increase in theconcentration of the complex can be achieved by increasing theprobability that the two molecules form a complex by binding a compoundto one of them. A compound that binds with high affinity to one of themolecules in the complex can increase the probability that a complexforms between the molecules if the compound increases the likelihoodthat amino acids between the two molecules can form favorableinteractions with one another.

The term “abnormal condition” refers to a function in an organism'scells or tissue that deviate from a normal function in the cells ortissue of that organism. In the context of the invention, an abnormalcondition is associated with an aberration in a signal transductionpathway involving FRS2. Abnormal conditions can be associated with cellproliferation. Cell proliferative disorders include cancers such asfibrotic and mesangial disorders, abnormal angiogenesis andvasculogenesis, slow wound healing rates, psoriasis, diabetes mellitus,and inflammation. Abnormal conditions can also be associated with celldifferentiation. Cell differentiation disorders include, but are notlimited to neurodegenerative disorders, slow wound healing rates, andslow tissue grafting healing rates.

The abnormal condition can be diagnosed, prevented, or treated when theorganism's cells exist within the organism or outside of the organism.Cells existing outside the organism can be maintained or grown in cellculture dishes. For cells harbored within the organism, many techniquesexist in the art to administer compounds, including (but not limited to)oral, parenteral, dermal, and injection applications. For cells outsideof the patient, multiple techniques exist in the art to administer thecompounds, including (but not limited to) cell microinjectiontechniques, transformation techniques, and carrier techniques.

The term “signal transduction pathway” refers to the molecules thatpropagate an extracellular signal through the cell membrane to become anintracellular signal. This signal can then stimulate a cellularresponse. The polypeptide molecules involved in signal transductionprocesses are typically receptor and non-receptor protein kinases,receptor and non-receptor protein phosphatases, and transcriptionfactors.

The term “aberration”, in conjunction with a signal transductionprocess, refers to a FRS2 protein or other protein involved in a signaltransduction pathway involving FRS2 that is over- or under-expressed inan organism, mutated such that its catalytic activity is lower or higherthan the wild-type molecule, mutated such that it can no longer interactwith a binding partner, is no longer modified by another protein kinaseor protein phosphatase, or no longer interacts with a binding partner.

The term “detecting a change in interactions”, in the context of theinvention, defines a method of determining whether a compound enhancesor blocks the interaction between FRS2 and natural binding partners.Multiple methods exist within the art that can detect a complex formedbetween FRS2 and natural binding partners. One such method is disclosedherein by example in relation to the interaction formed between FRS2 andGrb-2.

The interaction between FRS2 and natural binding partners can also bedetected by a difference in a cell morphology. Differences in cellmorphology include growth rates and differentiation rates of cells.These phenomena are simply measured by methods in the art. These methodstypically involve observing the number of cells or the appearance ofcells under a microscope with respect to time (days).

The method can utilize any of the molecules disclosed in the invention.These molecules include nucleic acid molecules encoding FRS2polypeptides, nucleic acid vectors, recombinant cells, polypeptides, orantibodies of the invention.

The method can be performed in vitro as well as in vivo. In vivoapplications include introducing a group of cells to an organism andthen determining the effect of a compound administered to the organismon the state of the organism as well as the introduced cells. The artcontains multiple methods of introducing a group of cells to an organismas well as methods of administering a compounds to an organism. Theorganism is preferably an animal such as a frog, more preferably amouse, rat, or rabbit, and most preferably a monkey, ape, or human.

Another aspect of the invention relates to a method of diagnosing anabnormal condition associated with cell proliferation or celldifferentiation in an organism. The abnormal condition can be associatedwith an aberration in a signal transduction pathway characterized by aninteraction between a FRS2 polypeptide and a natural binding partner.The method comprises of the step of detecting an abnormal interaction.

The term “detecting an abnormal interaction” defines a method ofidentifying a FRS2 molecule with an aberration in its activity.Detection is accomplished by using an antibody or antibody fragment ofthe invention, a nucleic acid probe of the invention, or a compound ofthe invention.

Techniques used in the art that incorporate this method include invitro, in vivo, and in situ hybridization techniques. These techniquesutilize nucleic acid probes of the invention.

A preferred embodiment of the invention is that the diagnosis methodrelates to an organism that is a mammal.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1A is the amino acid sequence of the FRS2 protein isolated from NIH3T3 cells. Four tryptic peptides are underlined in the amino acidsequence. The myristylation sequence is underlined with a hatched lineat the N-terminus. The portion of the FRS2 protein corresponding to thephosphotyrosine binding domain (PTB domain) is boxed in the figure.Putative SH2 binding regions of FRS2 are indicated in bold.

FIG. 1B aligns the sequence of the PTB domain of another adaptorprotein, IRS-1, with that of FRS2. Secondary structural elements(including α-helices and β-sheets) are boxed. Vertical lines reportidentical amino acids shared between the two proteins. Only twocontiguous amino acids are identical within the two proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part upon the isolation andcharacterization of nucleic acid molecules encoding an adaptor proteindesignated FRS2 that links protein kinases to activating proteins incells. The invention also relates to nucleic acid molecules encodingportions of the FRS2 polypeptide, nucleic acid molecules encoding atleast one FRS2 functional portion, nucleic acid vectors harboring suchnucleic acid molecules, recombinant cells containing such nucleic acidvectors, purified polypeptides encoded by such nucleic acid molecules,antibodies to such polypeptides, and methods of identifying compoundsthat enhance or block interactions of FRS2 with natural bindingpartners. Also disclosed are methods for diagnosing abnormal conditionsin an organism with FRS2 related molecules or compounds.

Experiments reported herein by example suggest that FRS2 links FGFR tothe activating Ras/MAPK pathway via Grb-2/Sos. Observations in theliterature underscore the importance of the FGFR-FRS2 interaction asalterations in FGFR function lead to diseased states in organisms.

A number of mutations in FGFR1 and FGFR2 lead to severe craniofacialabnormalities and characteristic abnormalities in the foot, thumb andtoes. These mutations are responsible for Pfeiffer, Jackson-Weiss andCrouzon syndromes (Muenke et al., 1994, Nature Genetics 8:269-274; Jabset al., 1994, Nature Genetics 8:275-279; Rutland et al., 1995, NatureGenetics 9:173-176). Experiments with mice genetically deficient inFGFR1 have confirmed the central role of this receptor in embryonicgrowth and axial gastrulation (Yamaguchi et al., 1994, Genes&Dev.8:3032-3044; Deng et al., 1994, Genes&Dev. 8:3045-3057).

Mutations have also been identified in FGFR3 that lead to skeletaldysplasias and hypochondroplasia (Tavormina et al., 1995, Hum. Mol.Genetics 4:2175-2177; Bellus et al., 1995, Nature Genetics 10:357-359).One mutation in FGFR3 is responsible for the most common form of humandwarfism (Shiang et al., 1994, Cell 78:335-342), apparently resultingfrom a gain-of-function mutation that leads to constitutive activationof the intrinsic protein kinase activity of the FGF receptor (Websterand Donoghue, 1996, EMBO 15:520-527). Inactivation of the FGFR3 gene inmice causes severe bone dysplasia with enhanced bone growth (Deng etal., 1996, Genes&Dev. 8:3045-3057).

Loss of function mutations identified in the C. elegans homologue of theFGF receptor also lead to severe developmental disorders (DeVore et al.,1995, Cell 83:611-620). Therefore, FRS2 is a drug design target forthese diseases. In addition, FRS2 is a drug design target for otherabnormal conditions in organisms since FGFR is an important regulatorymolecule in cell proliferation and differentiation pathways.

Various other features and aspects of the invention are: nucleic acidmolecules encoding a FRS2 polypeptide; recombinant DNA techniques tomanipulate nucleic acid molecules; nucleic acid probes for the detectionof FRS2; a probe-based method and kit for detecting FRS2 messages inother organisms; DNA constructs comprising a FRS2 nucleic acid moleculeand cells containing these constructs; methods of isolating, enrichingor purifying FRS2 polypeptides; FRS2 antibodies and hybridomas;antibody-based methods and kits for detecting FRS2; identification ofagents; isolation of compounds which interact with a FRS2 polypeptide;compositions of compounds that interact with FRS2 and FRS2 molecules;preparation of pharmaceutical formulations of compounds; modes ofadministration of compounds to organisms; purification and production ofcomplexes; derivatives of complexes; antibodies to complexes; disruptionof FRS2 protein complexes; transgenic animals containing FRS2 nucleicacid constructs; antisense and ribozyme approaches, gene therapy; andevaluation of disorders. Those skilled in the art appreciate that anymodifications made to a complex can be manifested in the modification ofany of the molecules in that complex. Thus, the invention includes anymodifications to nucleic acid molecules, polypeptides, antibodies, orcompounds in a complex. All of these aspects and features are explainedin detail with respect to PYK-2 in PCT publication WO 96/18738, which isincorporated herein by reference in its entirety, including anydrawings. Those skilled in the art will readily appreciate that suchdescriptions can be easily adapted to FRS2 as well, and is equallyapplicable to the present invention.

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention. The examplesbelow demonstrate the isolation, and characterization of the novelprotein FRS2. Materials and methods utilized in the experimentsdisclosed in the examples are as follows:

Cell lines: PC12 cells (Spivak-Kroizman et al., 1994, J. Biol. Chem.269:14419-14423), and L6 myoblasts (Mohammadi et al., 1992, Nature358:684-684) expressing FGFR1 were previously described. HER14 cells areNIH 3T3 cells that overexpress the human EGF-receptor (EGFR) (Honeggeret al., 1987, Cell 51:199-209)

Antibodies and GST-fusion proteins: Anti-Grb2 (N-SH3 domain), anti-Sosl,antiphosphotyrosine (anti-pY), anti-Shc, and anti-FGFR were previouslydescribed (Lowenstein et al., 1992, Cell 70:431-442; Spivak-Kroizman etal., 1994, J. Biol. Chem. 269:14419-14423). Anti-EGFR (Honegger et al.,1987, Cell 51:199-209) and anti-GST (Batzer et al., 1994, Mol. Cell.Biol. 14:5192-5201) antibodies were also previously described. Anti-FRS2antibodies were generated against a GST fusion protein containing theC-terminal portion of the protein (amino acids 400-508 of FRS2). A GSTfusion protein of Grb2, as well as fusion proteins of several Grb2mutants, GST-Grb2-SH2, GST-Grb2(P49L), GSTGrb2 FLVR (R86K), (Lowensteinet al., 1992, Cell 70:431-442; Clark et al., 1992, Nature 356:340-344;Skolnik et al., 1993, Science 260:1953-1955) were generated aspreviously described.

Immunoprecipitation and immunoblotting analysis: Transfected cellsexpressing either FGFR1 or EGFR were starved overnight: L6 cells ingrowth medium containing 0.5% fetal calf serum (FCS), and PC12 cells inmedium containing 0.5% FCS and 0.5% horse serum. Starved cells weretreated with 5 nM aFGF together with 5 μg/ml heparin, or with 16 nM EGF,and were incubated at 37° C. for 5 minutes, or for the indicated times.The cells were lysed and subjected to immunoprecipitation andimmunoblotting analysis as previously described (Spivak-Kroizman et al.,1994, J. Biol. Chem. 269:14419-14423).

Purification of FRS2: FRS2 was purified from NIH 3T3 cells that had beenstimulated as previously described (Spivak-Kroizman et al., 1994, J.Biol. Chem. 269:14419-14423) with aFGF and heparin. Cells were lysed,and the lysate was mixed with a Grb2-SH2 Affigel-10 column (10 mg SH2protein was cross linked to 1 ml Affigel 10, Bio-Rad) for 3 hours at 4°C. The column was washed three times with HNTG (20 mM Hepes, 150 mMNaCl, 1% Triton X-100, 10% glycerol), and proteins were eluted with 10mM Tris (pH 7.5) containing 1% SDS and 2.5% (v/v) β mercaptoethanol. Thecolumn was incubated with the elution buffer for 10 min. at 100° C. Theeluate was next diluted 50 fold with HNTG and the diluted protein wasmixed with affinity-purified antiphosphotyrosine antibodies (anti-P-Tyr)bound to protein-A sepharose for 5 hours at 4° C. FRS2 was eluted fromthe antibody column with 100 mM phenyl-phosphate in 10 mM Tris pH 7.5.The sample was then applied to an 8% SDS gel, transferred to anitrocellulose filter and visualized by staining with Ponceau S.

Protein sequencing: The band corresponding to FRS2 was excised from thenitrocellulose filter. The nitrocellulose was cut into 2 cm squarepieces, wetted with methanol, then reduced with 200 ul of 10 mM DTT, 200mM Tris pH 9.2, 5 mM EDTA at 50° C. for one hour and alkylated by theaddition of 1/10 volume of 250 mM 4-vinyl pyridine in acetonitrile for30 min. at room temperature. The nitrocellulose was then washed fivetimes with 10% acetonitrile in water, 50 μl of 100 mM Tris pH 8.2, 1%octylglucoside, 10% acetonitrile containing 200 ng of modified porcinetrypsin (Promega) was then added, and the sample was incubated 18 hrs at37° C. with constant shaking. The supernatant containing peptidesreleased from the nitrocellulose was injected directly onto reversedphase HPLC for peptide separation. A 1×150 mm Reliasil C-18 column wasused in a Michrom HPLC. Solvents were A=0.1% TFA in acetonitrile/water(3:97), B=0.9% acetonitrile/water (97:3). The column was washed with 5%B until a flat baseline is obtained, and was then eluted with a 5 to 65%B gradient over 60 minutes. Monitoring was at 214 nm, peaks werecollected into a deep well polypropylene microliter plate and storedfrozen. Sequencing was performed with an Applied Biosystems 494 usingstandard reagents and programs from the manufacturer.

cDNA cloning of FRS2: A pair of degenerate primers was synthesized basedupon the amino acid sequence of tryptic peptide #2 (VYENINGLSIPSASGV).PCR was performed with these two primers using cDNA prepared from mRNAisolated from 3T3 cells. The 60 base pair long product of this reactionwas sequenced and found to have the correct amino acid sequence. Asecond round of PCR was then performed with the same cDNA using oneprimer chosen from the sequence of the initial 60 bp reaction product,and a second degenerate primer based upon the sequence of trypticpeptide #1 of FRS2 (FVLGPTPVQK); this reaction gave a 170 base pairproduct. A third round of PCR was performed with one primer from this170 base pair product and a T3 primer from the Bluescript vector. The1.2 kb product of this reaction contained the sequence of peptide #1.Finally, the 1.2 kb fragment was used as a probe for screening a λ cDNAlibrary generated from Swiss 3T3 cells (Stratagene). Two phage clones ofλp90-1 and λp90-2 were isolated and further analyzed. Determination ofthe deduced amino acid sequences of these two clones revealed a longopen reading frame (ORF) that contained the sequences of the fourtryptic peptides that were isolated from purified FRS2.

Transient expression in 293 cells: 293 cells were grown to 70%confluency on 10 cm tissue culture dishes. Cells were transientlytransfected with various expression vectors (as indicated in thefigures) using the lipofectamine reagent (Gibco BRL). Empty expressionvectors were used to adjust the amount of DNA transfected to each plateto a constant of 10 μg.

[³H] myristic acid labeling: Subconfluent 293 cells were transfectedwith wild tp FRS2 or the G2A mutant; 36 hours after transfection, cellswere washed twice with DMEM supplemented with 2% dialysed FCS and wereincubated for 3 hours at 37° C. in the same medium to which 100 μCi/mL[³H] myristic acid (Amersham) was added. Cells were then lysed andimmunoprecipitated with anti-FRS2 antibodies. The immunoprecipitateswere separated by 8% SDS-PAGE, and the gel was treated with an ENTENSIFYsolution (Dupont) according to the manufacture's protocol. The gel wasdried and analyzed by autoradiography. As a control for normalizing theamount of protein loaded, a 10% of each immunoprecipitate wastransferred to a nitrocellulose filter and immunoblotted with anti-FRS2antibodies.

Immunofluorescence analysis: HeLa cells were transiently transfectedwith the HA-tagged construct of FRS2 or its G2A mutant. 24-48 hoursafter transfection the cells were plated on glass cover-slips coatedwith poly-L-lysine. 16 hours later, the cells were fixed with 4%paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 in PBSfor 20 minutes at room temperature. Cells were treated with a blockingsolution that contain 5% goat serum and 5% horse serum (VectorLaboratories, CA), stained with anti HA-antibody and secondaryfluorescein-conjugated goat anti-mouse antibodies.

Generation of PC12 cells stably expressing FRS2: FRS2 cDNA was clonedinto the LXSN expression vector, and a high titer stock of virus wasproduced as previously described. Rozakis-Adcock et al., 1992, Nature360:689-692. Parental cells were infected with a virus that combinedwild type FRS2 and the neomycin resistant gene and were selected for oneweek in medium supplemented with Geneticin (500 μg/ml). PC12 cells thatexpress Ras (N17) were infected with FRS2 expression virus which containhistidinol dehydrogenase as a selection marker. Cells were then selectedwith histidinol (800 μg/ml) for two weeks. Pools of selected cultureswere used in the studies. Expression of Ras (NI7) was induced byovernight treatment with 2 μM dexamethasone.

Analysis of PC 12 cells differentiation: PC12 cells infected with anFRS2 virus were seeded at a density of 10⁵ cells per 60 mm tissueculture dish. Cells were grown in DMEM containing 10% fetal calf serumand 10% horse serum for 24 hours. The medium was supplemented with aFGF(2.5 nM) and heparin (5 μg/ml) for an additional 48 hours. PC12 cellsthat coexpress FRS2 and Ras (N17) were treated in addition with 2 μMdexamethasone. Neurite outgrowth was quantitated by scoring randomgroups of 200 cells for the length of their neurites and determining theaverage length per cell for every treatment.

Example 1

SH2 Domain of Grb2 Binds to FRS2

Grb-2 contains one SH2 domain flanked by two SH3 domains. Lowenstein etal., 1992, Cell 70:431-442. Since tyrosine phosphorylated FRS2 binds toGrb2, its ability to interact with the SH2 domain alone was tested. Aglutathione-S-transferase (GST)-fusion protein of the Grb2 SH2 domainwas used to precipitate associating proteins from lysates ofFGF-stimulated cells. Analysis with anti-pY antibodies showed that both,tyrosine-phosphorylated Shc and FRS2 were associated with the Grb2 SH2domain in an FGF-dependent manner. By contrast, a GST fusion proteinwith a mutated, binding-defective form of the Grb2 SH2 (R86K) failed toprecipitate either protein.

The far-Western technique was applied to determine whether theinteraction between FRS2 and the SH2 domain of Grb2 was direct. Lysatesfrom FGF-stimulated or unstimulated cells were immunoprecipitated witheither anti-Grb2 or anti-pY antibodies, and analyzed by blotting withGST (as a control), GST-Grb2, or GST-Grb2 (P49L), a point mutant in theN-terminal SH3 domain corresponding to the Sem-5 loss-of-function allele(Clark et al., 1992, Nature 359:340-344. Both GST-Grb2 and the form withthe mutated SH3 domain bound directly to tyrosine-phosphorylated FRS2(data not shown), providing further confirmation of a direct interactioninvolving the Grb2 SH2 domain.

Example 2

Grb2/FRS2 Complex is Bound to Sos in FGF-Stimulated Cells

Grb2 binds to the guanine-nucleotide releasing factor Sos1 through itstwo SH3 domains. Schlessinger, 1994, Curr. Opin. Gen. Dev. Biol.4:25-30. Experiments were performed to determine whether the complexformed between FRS2 and Grb2 also interacts with Sos1. Lysates ofaFGF-stimulated or unstimulated cells were immunoprecipitated withantibodies against FGFR1, Grb2, or Sos1. The immunoprecipitates wereseparated by SDS-PAGE, and immunoblotted with anti-phosphotyrosineantibodies. Tyrosine-phosphorylated FRS2 was seen to be associated withboth Grb2 and Sos1 in FGF-stimulated cells, but was not detected inanti-FGFR1 (or anti-Shc) immunoprecipitates. This experiment indicatedthat a ternary complex, composed of Grb2, Sos1 and FRS2 was formed inresponse to aFGF stimulation. Far-western blotting experiments alsoconfirmed the existence of a complex containing FRS2, Grb2, and Sos1.The experiments suggested that association between Grb2 and FRS2 wasmediated by the SH2 domain of Grb2, and was dependent upon FGFstimulation. The association between Sos1 and Grb2 was constitutive andmediated by the SH3 domains.

Example 3

Purification of FRS2

The experiments in Example 1 showed that the SH2 domain of Grb2 binds totyrosine phosphorylated FRS2. This information was used to develop anaffinity chromatography method for the purification of FRS2. NIH 3T3cells were stimulated with aFGF, lysed, and the cell lysate was appliedto an affigel affinity matrix containing immobilized Grb2 SH2 domain.The tyrosine phosphorylated FRS2 bound the SH2 affigel matrix and wasreleased from the matrix by boiling in the presence of SDS and reducingagents. The FRS2 containing sample was then subjected to a secondaffinity chromatography purification step using anti-phosphotyrosineantibodies. Tyrosine phosphorylated FRS2 was bound toanti-phosphotyrosine antibodies, and the complex was applied to aprotein A-sepharose column. Phosphorylated FRS2 was then released fromthe column using 100 mM phenyl phosphate.

The eluted protein was analyzed by SDS-PAGE. After transfer tonitrocellulose, Ponceau-S staining revealed a doublet of apparentmolecular weight of 92/95 kDa, corresponding to FRS2. The purified FRS2protein band was excised from the filter, digested with trypsin, and thetryptic peptides were resolved by reverse phase HPLC. The amino acidsequences of four tryptic peptides were determined using a solid phasemicrosequencer.

Example 4

cDNA Cloning of FRS2

Peptide sequences from FRS2 were used to design oligonucleotides foramplification by polymerase chain reaction (PCR) of cDNA prepared frommRNA isolated from NIH-3T3 cells. A 1.2 kb PCR product contained thesequences of proteolytic peptide #1 (FVLGPTPVQK) and peptide #2(VYENINGLSIPSASGV) from FRS2. The 1.2 kb PCR product was then used as aprobe for screening a cDNA library from NIH-3T3 cells. Two overlappingclones, λp90-1 and λp90-2, were isolated. Determination of thenucleotide sequences of the two clones demonstrated that λp90-2contained the sequences of all four tryptic peptides isolated from FRS2and that λp90-2 represents a full-length cDNA clone of FRS2.

The deduced amino acid sequence of FRS2 determined from clone λp90-2 ispresented in FIG. 1A. The coding sequence of FRS2 begins at nucleotidenumber 308. The first methionine is within a Kozak consensus sequence,and is followed by an open reading frame (ORF) of 1527 base pairs,ending with a stop codon at nucleotide 1834. This ORF encodes a proteincontaining 508 amino acids with a predicated molecular mass of 56800daltons. The sequence of FRS2 contains a consensus myristylationsequence (MGXXXS/T) at the amino terminus of the molecule MGSCCS. Resh,1994, Cell 76:411-413. In addition, FRS2 contains a stretch of 120 aminoacids (residues 11 to 139) with 29% sequence identity to the PTB(phosphotyrosine binding) domain of IRS1 (FIG. 1B). Sun et al., 1991,Nature 352:7377. It has been shown that the PTB domain of IRS1 binds toa tyrosine-phosphorylated NPXYp sequence in the juxtamembrane region ofthe insulin receptor. White et al., 1988, Cell 54:641-649. FRS2 alsocontains two potential Grb2 binding sites (NYEN and NYVN) (FIG. 1B).

The tissue expression pattern of FRS2 were examined by Northern blotanalysis of mRNA isolated from adult mouse tissues. FRS2 wasubiquitously expressed and most abundant in brain, kidney, lung, ovaryand testis. Polyclonal antibodies were raised in rabbits against aC-terminal portion of FRS2 expressed in E.coli as a GST fusion proteinto characterize the FRS2 protein. The anti-FRS2 antibodies precipitateda protein from NIH-3T3 cells as well as from PC12 cells that migrates inSDS-PAGE as a doublet of 92-95 kDa. The discrepancy between thepredicted molecular weight of FRS2 (56,800 daltons) versus its migrationin SDS gels may result in part from post-translational modifications.

The association between Grb2 and FRS2 in lysates from NIH-3T3 cellsusing anti-FRS2 antibodies were also examined. NIH-3T3 cells werestimulated with aFGF, and lysates prepared from unstimulated orstimulated cells were then subjected to immunoprecipitation withantibodies against Grb2, FRS2, Sos1, Shc or FGFR1, followed byimmunoblotting with several different antibodies. This experiment showedligand-dependent association of Grb2 with tyrosine phosphorylated FRS2in aFGF stimulated cells. Similarly, Sos1 was found to be associatedwith FRS2 only in aFGF-stimulated cells. Interestingly, more pronouncedco-immunoprecipitation of Grb2 and Sos1 was detected in lysates of aFGFstimulated cells, suggesting that the interaction between Grb2 andtyrosine phosphorylated FRS2 may stabilize complex formation betweenGrb2 and Sos1. Association between FRS2 and FGFR1 or between FRS2 andShc in FGF-stimulated or unstimulated cells was not detected. Similarly,the association between FRS2 and other signaling molecules such as Nck,phospholipase Cλ, or p85, the regulatory subunit of PI-3 kinases werealso not detected. Taken together, these experiments demonstrate thataFGF stimulation leads to tyrosine phosphorylation of FRS2, which inturn binds to Grb2/Sos1 forming an FGF-dependent ternary complex.

Example 5

FRS2 is Myristylated and Targeted to the Cell Membrane

The amino terminus of FRS2 contains a putative myristylation sequencefollowed by a phosphotyrosine binding domain (PTB domain) similar to thePTB domain of IRS1. Resh, 1994, Cell 76:411-413. The role of theputative myristylation sequence was examined by generation of a pointmutant (G2A) in which a key glycine residue in the consensus sequencewas replaced by alanine. Expression vectors that directed the synthesisof wild type FRS2 or the mutant G2A were transiently expressed in human293 cells. The transfected cells were labeled with [³H] myristic acid,lysed, subjected to immunoprecipitation with anti-FRS2 antibodies, andanalyzed by 8% SDS-PAGE and autoradiography. Incorporation of [³H]myristic acid could be detected in wild type FRS2 but not in the G2Apoint mutant.

The cellular distribution of FRS2 was analyzed in transfected cells.HeLa cells were transiently transfected with an expression vector thatdirects the synthesis of an HA-tagged form of wild type FRS2 or thepoint mutant (G2A). Transfected cells were permeabilized and labeledwith fluorescein labeled anti-HA antibodies. Visualization by confocalfluorescence microscopy demonstrated that, while wild type FRS2 wasprimarily associated with the cell membrane, the G2A mutant wasdistributed throughout the cytoplasm. This experiment demonstrated thatwild type FRS2 was targeted to the cell membrane and that myristylationwas required for this localization.

A cellular fractionation procedure was used to study the distribution ofwild type FRS2 and the G2A mutant. Particulate and soluble fractionswere prepared from untreated or aFGF-treated cells and FRS2 wasimmunoprecipitated from both cellular fractions. FGFR1 was used as amarker to ascertain that the fractionation protocol correctly separatedcytosolic from membrane bound proteins. FGFR1 was identified exclusivelyin the particulate fraction and was phosphorylated on tyrosine residuesin aFGF stimulated cells. Endogenous FRS2 was found to be associatedexclusively with the particulate fraction, and aFGF stimulation led totyrosine phosphorylation of FRS2 and binding to Grb2. By contrast, thenon-myristylated mutant (G2A) of FRS2 was found in the soluble fractionand was not tyrosine phosphorylated in FGF stimulated cells. Theelectrophoretic mobility of the non-myristylated form was altered inFGF-stimulated cells, suggesting that aFGF may induce Ser/Thrphosphorylation of the mutant FRS2 protein. The studies show that FRS2was myristylated, and that myristylation was essential for its targetingto the cell membrane, for tyrosine phosphorylation of FRS2 and forrecruitment of Grb2.

Example 6

Activation of the MAPK Pathway by Overexpression of FRS2

The ability of FRS2 to recruit Grb2 to the cell membrane in response toaFGF stimulation raised the possibility that its physiological role isto link FGF receptor activation to the Ras/MAP kinase signaling pathway.To explore this possibility, increasing concentrations of FRS2 wastransiently overexpressed together with expression of a constant amountof the FGF receptor and an HA-tagged ERK1, in 293 cells. The activity ofERK1 was measured in an immunocomplex assay (Dikic et al., 1996, J.Biol. Chem. 270:15125-15129) using myelin basic protein (MBP) as asubstrate. Overexpression of FRS2 lead to a proportional increase intyrosine phosphorylation of MAP kinase and phosphorylation of MBP.

While overexpression of wild type FRS2 led to strong activation of MAPkinase, overexpression of the non-myristylated mutant led to adrastically reduced MAP kinase activation. MAP kinase activation inducedby overexpression of the non-myristylated mutant was reduced by nearly70% as compared to activation of MAP kinase induced by overexpression ofwild type FRS2, after subtraction of the background activation inducedby endogenous FRS2 molecules that are expressed in these cells. Unlikewild-type FRS2 which was tyrosine phosphorylated and hence bound toGrb2, the non-myristylated FRS2 mutant was not tyrosine phosphorylatedin response to FGFR activation.

To examine the role of Ras in this process the effect of FRS2overexpression on MAP kinase activity was analyzed in the presence of adominant interfering mutant of Ras(N17). This experiment showed thatexpression of the dominant interfering mutant of Ras efficiently blockedFRS2-induced MAP kinase activation. However, the tyrosinephosphorylation of FRS2 and its association with Grb2 were not affectedby the expression of the dominant interfering Ras(N17) mutant. Theseexperiments therefore demonstrated that FRS2 functioned as a linkbetween activated FGF receptors and the Ras/MAP kinase cascade, and thatFRS2 acted upstream of Ras in this pathway.

Example 7

Overexpresion of FRS2 Promotes Neurite Outgrowth in PC12 Cells

PC12 cells were stably transfected with expression vectors that directedthe synthesis of either wild type FRS2 or the non-myristylated G2Amutant. The cells were incubated with a low concentration of aFGF andheparin that induced weak, barely detectable, neurite outgrowth in theparental PC12 cells. The experiment demonstrated that overexpression ofFRS2 led to strong potentiation of aFGF-induced neurite outgrowth inthese PC12 cells. By contrast, expression of the non-myristylated FRS2mutant did not influence aFGF-induced neurite outgrowth in thetransfected cells.

It was noteworthy that the PC12 cell line used in this study expressedendogenous FRS2 and low levels of endogenous FGF receptors. Therefore,aFGF-stimulation promoted flattening, adhesion, and weak neuriteoutgrowth of control cells. Immunoprecipition and immunoblottingexperiments confirmed that endogenous and transfected wild type FRS2were both tyrosine phosphorylated, while the non-myristylated FRS2mutant is not tyrosine phosphorylated in response to aFGF stimulation.

The role of Ras in FRS2-induced neurite outgrowth was tested byconditional expression in PC12 cells of a dexamethasone-inducibledominant interfering mutant of Ras(N17). Overexpression of Ras(N17)affected neither the expression of FRS2 nor tyrosine phosphorylation ofFRS2. However, FRS2-induced neurite outgrowth was totally blocked byoverexpression of Ras(N17), indicating that it is dependent upon Rasactivation.

Although certain embodiments and examples have been used to describe thepresent invention, it will be apparent to those skilled in the art thatchanges to the embodiments and examples shown may be made withoutdeparting from the scope or spirit of the invention.

Those references not previously incorporated herein by reference,including both patent and non-patent references, are expresslyincorporated herein by reference for all purposes.

Other embodiments are encompassed by the following claims.

What is claimed is:
 1. An isolated, enriched or purified polypeptide,comprising the amino acid sequence set forth in SEQ ID NO:9.
 2. Anisolated, enriched, or purified polypeptide, wherein said polypeptidecomprises at least 100 contiguous amino acid residues of the amino acidsequence set forth in SEQ ID NO:9.
 3. The polypeptide according to claim2, wherein said polypeptide comprises at least 141 contiguous amino acidresidues of the amino acid sequence set forth in SEQ ID NO:9.
 4. Thepolypeptide according to claim 2, wherein said polypeptide comprises atleast 200 contiguous amino acid residues of the amino acid sequence setforth in SEQ ID NO:9.
 5. The polypeptide according to claim 2, furthercomprising a second polypeptide, said second polypeptide fused to saidpolypeptide.
 6. The polypeptide according to claim 5, wherein saidsecond polypeptide is at least one selected from the group consisting ofglutathione-S-transferase, hemagglutinin, and maltose-binding protein,or a fragment of any one of said second polypeptides.
 7. An isolated,enriched or purified polypeptide, comprising an amino acid sequencethat: (a) differs from the sequence set forth in SEQ ID NO:9 in that itlacks at least one, but not all, of the following segments of amino acidresidues: 1-10, 11-152 or 153-508; or (b) has the amino acid sequenceset forth from amino acid residues 1-10, 11-152 or 153-508 of SEQ IDNO:9.
 8. An isolated, enriched or purified polypeptide, comprising anamino acid sequence that differs from the sequence set forth in SEQ IDNO:9 in that it lacks at least one, but not all, of the domains selectedfrom the group consisting of a myristylation region, a phosphotyrosinebinding domain and a C-terminal region.
 9. The polypeptide according toclaim 7 or claim 8, further comprising a second polypeptide, whereinsaid second polypeptide is fused to said amino acid sequence.
 10. Thepolypeptide according to claim 9, wherein said second polypeptide is atleast one selected from the group consisting ofglutathione-S-transferase, hemagglutinin, and maltose-binding protein,or a fragment of any one of said polypeptides.